Antibiotics called `chloropolysporins B and C`, a process for their preparation, and their therapeutic and veterinary use

ABSTRACT

Novel compounds, called chloropolysporins B and C, and salts thereof have antibiotic properties and would appear to be members of the class of glycopeptide antibiotics containing chlorine. They may be produced by cultivating a suitable strain of miroorganism of the genus Micropolyspora, especially Micropolyspora sp. SANK 60983 (FERM BP-538). They may be combined with conventional pharmaceutically acceptable carriers or diluents for therapeutic use or incorporated into edible excipients, such as feed or water, for use as a growth-promoting agent, especially for farm animals.

This application is a continuation of application Ser. No. 06/813,617,filed Dec. 26, 1985 now abandoned.

BACKGROUND TO THE INVENTION

The present invention relates to new antibiotics, which we have called"chloropolysporins B and C", to a process for their preparation by thecultivation of a microorganism and to their use, both therapeutic, inthe treatment and prophylaxis of infections caused by bacteria, and as agrowth-promoting agent for animals.

As resistance to conventional antibiotics becomes increasinglyestablished in common strains of pathogenic bacteria, the need for awider variety of antibiotics for use in the fight against such bacteriabecomes ever more crucial. Moreover, various antibiotics, for examplechloramphenicol, aureomycin, vancomycin and avoparcin, have beenadministered, or have been proposed for administration, to poultry andother farm animals, including the ruminants and pigs, for theprophylaxis of disease or to promote growth or milk production. However,an inherent disadvantage of the use of antibiotics in this way is thatthere is some risk that traces of the antibiotics or of metabolicproducts thereof may be found in animal products intended for humanconsumption (such as eggs, milk or meat); the alleged dangers of suchresidues are increasingly criticized by some sections of the community.There is, accordingly, a considerable desire amongst farmers for anantibiotic substance which will leave the desired growth-promotingeffect but which will leave no or no significant residues in animalproducts.

In U.S. patent application Ser. No. 627,439, filed July 3rd 1984, whichissued as U.S. Pat. No. 4,557,933, on Dec. 10, 1985 , assigned to thepresent assignees, there is disclosed an antibiotic, there referred toas "chloropolysporin", which was isolated from the culture medium of amicroorganism identified as Micropolyspora sp. SANK 60983.

We have now discovered that the same microorganism, and hence others ofthe genus Micropolyspora, produces a further two new antibioticsubstances that are highly effective against gram-positive bacteria andthat show considerable promise for use as growth-promoting agents inanimals. It is believed that these substances may have been present inthe chloropolysporin of the prior Application.

BRIEF SUMMARY OF INVENTION

The new antibiotic substances of the invention are called"chloropolysporin B" and "chloropolysporin C" and, since theirstructures have not been completely elucidated, they may becharacterized by their properties.

Chloropolysporin B, as its sulfate, is characterized by the properties:

(a) it takes the form of an amphoteric white powder, soluble in water;

(b) specific rotation: [α]²⁵ -64.5° (C=1.04, 0.1N aqueous hydrochloricacid, sodium D-line);

(c) elemental analysis: C, 48.33%; H, 5.05%; N, 5.48%; Cl, 5.11%; S,1.00%;

(d) on acid hydrolysis it yields: neutral saccharides: glucose, mannoseand rhamnose; amino acids: 3-chloro-4-hydroxyphenylglycine andN-methyl-p-hydroxyphenylglycine;

(e) ultraviolet absorption spectrum: as illustrated FIG. 1 of theaccompanying drawings, having an absorption maximum λ_(max) at 280nm(E_(1cm) =51) in a 0.1 N solution of hydrochloric acid, the absorbence,E, being measured at a concentration of 1% w/v;

(f) infrared absorption spectrum: the infrared absorption spectrum (νcm⁻¹) measured on a KBr disc is as shown in FIG. 2 of the accompanyingdrawings;

(g) nuclear magnetic resonance spectrum: the nuclear magnetic resonancespectrum (δ ppm), measured at 270 MHz in deuterated dimethyl sulfoxideusing tetramethylsilane as the internal standard, is as illustrated inFIG. 3 of the accompanying drawings;

(h) solubility: soluble in water and methanol, sparingly soluble inacetone, and insoluble in ethyl acetate, chloroform and benzene;

(i) color reactions: positive in Ninhydrin and Rydon-Smith reactions;

(j) thin layer chromatography: Rf value=0.65, using a cellulose sheet(Eastman) as adsorbent and a 15:10:3:12 by volume mixture of butanol,pyridine, acetic acid and water as the developing solvent;

(k) high voltage paper electrophoresis: using Toyo's filter paper No.51A in a 0.1M tris-hydrochloric acid buffer solution of pH 7.5 (3300volt/60 cm, 1 hour); the migration distance (detected by bioautographywith Bacillus subtilis PCI 219) from the origin to the cathode was 4 cm;

(l) molecular formula: C₈₃ H₈₉ O₃₄ N₈ Cl₃. 0.5H₂ SO₄. 10H₂ O;

(m) molecular weight: the molecular weight, measured by FAB-MS, was 1846(MH⁺, 1847).

"FAB-MS" is Fast Atom Bombardment Mass Spectroscopy. Chloropolysporin C,as its sulfate, may be characterized by the following properties:

(a) it takes the form of an amphoteric white powder, soluble in water;

(b) specific rotation: [α]²⁵ -64.4° (C=1.08, 0.1N aqueous hydrochloricacid, sodium D-line);

(c) elemental analysis: C, 50.53%; H, 4.69%; N, 6.14%; Cl, 5.62%; S,1.12%;

(d) on acid hydrolysis it yields: neutral saccharides: glucose andmannose; amino acids: 3-chloro-4-hydroxphenylglycine andN-methyl-p-hydroxyphenylglycine;

(e) ultraviolet absorption spectrum: as illustrated in FIG. 4 of theaccompanying drawings, having an absorption maximum λ_(max) at 280nm(E_(1cm) =57) in a 0.1 N solution of hydrochloric acid, the absorbence,E, being measured at a concentration of 1% w/v;

(f) infrared absorption spectrum: the infrared absorption spectrum(νcm⁻¹) measured on a KBr disc is as shown in FIG. 5 of the accompanyingdrawings;

(g) nuclear magnetic resonance spectrum: the nuclear magnetic resonancespectrum (δ ppm), measured at 400 MHz in deuterated dimethyl sulfoxideusing tetramethylsilane as the internal standard, is as illustrated inFIG. 6 of the accompanying drawings;

(h) solubility: soluble in water and methanol, sparingly soluble inacetone, and insoluble in ethyl acetate, chloroform and benzene;

(i) color reactions: positive in Ninhydrin and Rydon-Smith reactions;

(j) thin layer chromatography: Rf value=0.65, using a cellulose sheet(Eastman) as adsorbent and a 15:10:3:12 by volume mixture of butanol,pyridine, acetic acid and water as the developing solvent;

(k) molecular formula: C₇₇ H₇₉ O₃₀ N₈ Cl₃. 0.5H₂ SO₄. 5H₂ O;

(1) molecular weight: the molecular weight, measured by FAB-MS, was 1700(MH⁺, 1701).

The present invention provides these compounds for the first time in aform suitable for medicinal or veterinary use, free from nativeimpurities.

The invention also provides pharmaceutically acceptable salts of thesecompounds.

The invention also provides a process for producing chloropolysporin Bor chloropolysporin C and salts thereof by cultivating achloropolysporin B- or C-producing microorganism of the genusMicropolyspora in a culture medium therefor and isolatingchloropolysporin B or C or a salt thereof from the cultured broth.

The invention still further provides a pharmaceutical or veterinarycomposition comprising such chloropolysporin B or C or a salt thereof inadmixture with a pharmaceutical or veterinary carrier or diluent.

The invention still further provides a method for the treatment orprophylaxis of bacterial infections by administering suchchloropolysporin B or C or a salt thereof to an animal, which may behuman or non-human.

The invention still further provides a method of promoting the growth ofa farm animal by the oral administration of such chloropolysporin B or Cor a salt thereof to said animal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the ultraviolet absorption spectrum of chloropolysporin B ofthe invention;

FIG. 2 is the infrared absorption spectrum of chloropolysporin B of theinvention;

FIG. 3 is the NMR spectrum of chloropolysporin B of the invention;

FIG. 4 is the ultraviolet absorption spectrum of chloropolysporin C;

FIG. 5 is the infrared absorption spectrum of chloropolysporin C; and

FIG. 6 is the NMR spectrum of chloropolysporin C.

DETAILED DESCRIPTION OF INVENTION

The invention is based upon the discovery and isolation ofchloropolysporin B and chloropolysporin C and salts thereof, each freefrom contamination by other products of cell metabolism, and preferablyfree from each other.

Chloropolysporins B and C are produced by the cultivation of aMicropolyspora strain herein identified as Micropolyspora sp. SANK60983, which was isolated from a soil sample collected in TochigiPrefecture, Japan.

The microorganism, Micropolyspora SANK 60983, has the characteristicsdescribed hereafter and is as described in U.S. patent application Ser.No. 627,439, referred to above. These characteristics were determined bycultivation on various media prescribed by the ISP (InternationalStreptomyces Project) or with the media recommended by S.A. Waksman inVolume 2 of "The Actinomycetes", in all cases at a temperature of 28° C.

1. Morphological Characteristics

Strain SANK 60983 grows relatively well on various media. The aerialmycelium is hardly visible on almost all media but may occasionally bevisible on glycerol-asparagine agar or on potato extract-carrot extractagar. The aerial and vegetative mycelia bear, at the top and the middle,short chains of spores, normally from 1 to 20, although occasionallymore than 20, spores. No distinct fragmentation of the hyphae isobserved with the strain, although it may be observed during laterstages of the culture.

2. Culture Characteristics

Strain SANK 60983 can produce pale yellow, yellowish brown or yellowishgray colors. Aerial hyphae are not observed on most media, althoughwhite aerial hyphae are produced on some media. No soluble pigment isproduced. Table 1 shows the results obtained after cultivation for 14days at 28° C. on various standard culture media. The color names andnumbers used were assigned according to the "Guide to Color Standard", amanual published by Nippon Shikisai Kenkyusho, Tokyo, Japan.

                  TABLE 1                                                         ______________________________________                                                          Aerial             Soluble                                  Medium  Growth    Mycelium  Reverse  Pigment                                  ______________________________________                                        Yeast   Abundant, None      Yellowish-                                                                             None                                     extract-                                                                              raised,             brown                                             malt    wrinkled,           (8-8-8)                                           extract yellowish-                                                            agar    brown                                                                 (ISP 2) (8-8-8)                                                               Oatmeal Good,     None      Dull     None                                     agar    smooth,             yellow                                            (ISP 3) dull                (8-8-9)                                                   yellow                                                                        (8-8-9)                                                               Inorganic                                                                             Abundant, None      Yellowish-                                                                             None                                     salt-   smooth,             gray (2-9-10)                                     starch  yellowish-          to pale                                           agar    gray                yellowish-                                        (ISP 4) (2-9-10)            brown                                                     to pale             (6-8-9)                                                   yellowish-                                                                    brown                                                                         (6-8-9)                                                               Glycerol-                                                                             Good,     Poor,     Yellowish-                                                                             None                                     asparagine                                                                            wrinkled, white     brown                                             agar    yellowish-          (2-9-10)                                          (ISP 5) brown                                                                         (2-9-10)                                                              Peptone-                                                                              Moderate, None      Pale     None                                     yeast   smooth,             yellowish-                                        extract-                                                                              pale                brown                                             iron agar                                                                             yellowish-                                                            (ISP 6) brown               (4-8-9)                                                   (2-8-9)                                                               Tyrosine                                                                              Abundant, None      Dull     None                                     agar    raised,             yellow                                            (ISP 7) wrinkled,           (10-8-8)                                                  pale                                                                          yellowish-                                                                    brown                                                                         (14-8-9)                                                              Sucrose Abundant, None      Pale     None                                     nitrate raised,             yellowish-                                        agar    wrinkled,           brown                                                     pale                (4-8-8)                                                   yellow                                                                        (12-8-10)                                                             Glucose-                                                                              Moderate  None      Yellowish-                                                                             None                                     asparagine                                                                            smooth,             gray                                              agar    yellowish-          (2-9-10)                                                  gray                                                                          (2-9-10)                                                              Nutrient                                                                              Moderate  None      Pale     None                                     agar    smooth,             yellowish-                                        (Difco) pale                brown                                                     yellowish-          (6-8-9)                                                   brown                                                                         (6-8-9)                                                               Water   Poor,     None      Yellowish-                                                                             None                                     agar    smooth,             gray                                                      yellowish-          (1-9-10)                                                  gray                                                                          (1-9-10)                                                              Potato  Moderate  Poor,     Yellowish-                                                                             None                                     extract-                                                                              smooth,   white     gray                                              carrot  yellowish-          (2-9-10)                                          extract gray                                                                  agar    (2-9-10)                                                              ______________________________________                                    

3. Physiological Properties.

The physiological properties of strain SANK 60983 are shown in Table 2.

                  TABLE 2                                                         ______________________________________                                         Decomposition:   Adenine        -                                                              Casein         +                                                              Xanthine       -                                                              Hypoxanthine   +                                                              Urea           +                                            Hydrolysis of starch             ±                                         Liquefaction of gelatin          +                                            Coagulation of milk              -                                            Peptonization of milk            -                                            Reduction of nitrate             +                                            Secretion of                     +                                            deoxyribonuclease                                                             Melanin formation:                                                                              ISP 1          -                                                              ISP 6          -                                                              ISP 7          -                                            Acid production from:                                                                           Sodium Acetate -                                                              Sodium Succinate                                                                             -                                                              Sodium Citrate -                                                              Sodium Pyruvate                                                                              -                                                              Sodium Tartarate                                                                             -                                                               .sub.--D-Glucose                                                                            +                                                               .sub.--L-Arabinose                                                                          +                                                               .sub.--D-Xylose                                                                             +                                                              Inositol       +                                                               .sub.--D-Mannitol                                                                           +                                                               .sub.--D-Fructose                                                                           +                                                               .sub.--L-Rhamnose                                                                           +                                                              Sucrose        +                                                              Raffinose      ±                                         Utilization of     .sub.--D-Glucose                                                                            +                                            carbon sources:    .sub.--L-Arabinose                                                                          +                                                               .sub.-- D-Xylose                                                                            +                                                              Inositol       +                                                               .sub.--D-Mannitol                                                                           +                                                               .sub.--D-Fructose                                                                           +                                                               .sub.--L-Rhamnose                                                                           +                                                              Sucrose        +                                                              Raffinose      ±                                         Growth in NaCl:    3% w/v        +                                                               5% w/v        ±                                                            7% w/v        ±                                                            10% w/v       -                                            Range of growth   10° C.  -                                            temperature:      20° C.  +                                                              28° C.  +                                                              37° C.  +                                                              45° C.  -                                            ______________________________________                                    

In the above Table, "+" means positive, "-" means negative and "±" meansslightly positive.

Although coagulation and peptonization of milk are both reported asnegative, they may occasionally turn positive after long-term culture.

4. Whole Cell Components

Acid hydrolyzates of bacterial cells were assayed by paperchromatography, using the method of M.P. Lechevalier et al. ["TheActinomycetes Taxonomy", page 225 (1980)]. meso-Diaminopimelic acid,arabinose and galactose were found to be present in the cell walls,which are thus of Type IV, whilst the whole cell sugar pattern is ofType A. The characteristic acyl group of the cell wall was alsoinvestigated by the method of Uchida et al. [J. Gen. Appl. Microbiol,23,249 (1977)] and found to be the acetyl group.

None of the known genera of actinomycetes has been reported to becapable of forming spores in the middle of the hyphae. However, from acomparison of other characteristics, the new strain is clearly relatedto the genera Actinopolyspora, Saccharopolyspora, Pseudonocardia andMicropolyspora. However, both Actinopolyspora and Saccharopolysporaallow spores to grow only on the tips of aerial hyphae, and the formeris a highly halophilic genus, whilst the characteristic acyl group ofthe cell wall of the latter is the glycolyl group. For these reasons,the new strain SANK 60983 cannot be assigned to either of these genera.Although strains of the genus Pseudonocardia can grow spores on theaerial hyphae and on the vegetative mycelium, as does strain SANK 60983,the site of its growth takes place only at the tip of the hyphae and,moreover, its hyphae characteristically grow by budding; thus, strainSANK 60983 cannot be assigned to the genus Pseudonocardia.

The only difference between the genus Micropolyspora and strain SANK60983 is that the site of growth of spores of Micropolyspora is limitedto the tips of the hyphae, whereas that of SANK 60983 is at both the tipand the middle of the hyphae.

At the present time, when there has been virtually no discussion inlearned circles as to the implications for taxonomy of differences ofthis type, it would seem inappropriate to differentiate between generasolely on the basis of differences in the site of growth of theirspores. Accordingly, it seems most satisfactory to regard the strainSANK 60983 as representative of a new species of the genusMicropolyspora and it has, accordingly, been named Micropolyspora sp.SANK 60983. It should, however, be remembered that assignment of astrain of microorganism to any particular species, genus or even familyis largely a matter of consensus amongst those experienced in the studyof the particular class of microorganism involved and the originalassignment of a microorganism can be, and not infrequently is, changedafter wider discussion.

The strain SANK 60983 has been deposited with the Fermentation ResearchInstitute, Agency of Industrial Science and Technology, Ministry ofInternational Trade and Industry, Japan, on Mar. 10th 1983 under theaccession No. FERM P-6985 and was re-deposited in accordance with theconditions stipulated by the Budapest Treaty with said FermentationResearch Institute on June 8th 1984 under the accession No. FERM BP-538.

It has been established that strain SANK 60983 produceschloropolysporins B and C. However, as is well known, the properties ofmicroorganisms falling within the general category of the actinomycetescan vary considerably and such microorganisms can readily undergomutation, both through natural causes and as the result of induction byartificial means. Accordingly, the process of the present inventionembraces the use of any microorganism which can be classified within thegenus Micropolyspora and which shares with the strain SANK 60983 thecharacteristic ability to produce chloropolysporins B and C.

The cultivation of microorganisms of the genus Micropolyspora inaccordance with the present invention to produce chloropolysporins B andC can be performed under conditions conventionally employed for thecultivation of actinomycete species, preferably in a liquid culture, anddesirably with shaking or stirring and aeration. The nutrient mediumused for the cultivation is completely conventional and contains suchconstituents as are commonly used in the cultivation of theactinomycetes. Specifically, the medium should contain an assimilablecarbon source, suitable examples of which include glucose, maltose,sucrose, mannitol, molasses, glycerol, dextrin, starch, soybean oil andcottonseed oil; an assimilable nitrogen source, suitable examples ofwhich include soybean meal, peanut meal, cottonseed meal, fish meal,corn steep liquor, peptone, meat extract, pressed yeast, yeast extract,sodium nitrate, ammonium nitrate or ammonium sulfate; and one or moreinorganic salts, such as sodium chloride, phosphates, calcium carbonateand trace metal salts. Where cultivation is effected in a liquid medium,it is generally desirable to incorporate an anti-foaming agent (forexample silicone oil, vegetable oil or a suitable surfactant) in themedium.

The cultivation is suitably performed at a substantially neutral pHvalue and at a temperature of from 24° to 30° C., more preferably atabout 28° C.

The production of chloropolysporins B and C as cultivation proceeds maybe monitored by a variety of conventional microbiological assaytechniques for monitoring the production of antibiotics (when they areproduced by microbial culture) and which require little or noelaboration here. A suitable technique might be the paper disc-agardiffusion assay (using, for example, a paper disc of diameter about 8 mmproduced by Toyo Kagaku Sangyo Co., Ltd) and using, for example,Bacillus subtilis PCI 219 or Staphylococcus aureus FDA 209P JC-1 as thetest organism.

The amount of chloropolysporins B and C produced normally reaches amaximum after cultivation has proceeded for 55-70 hours and it isclearly desirable to separate the chloropolysporins from the culturemedium no later than the time when this maximum has been reached.However, this period may vary, depending upon the cultivation conditionsand techniques, and a shorter or longer period may be appropriate,depending upon the circumstances. The correct cultivation time mayreadily be assessed for every case by routine experiment, using suitablemonitoring techniques, e.g. as described above.

Chloropolysporins B and C are mainly released into the liquid portion ofthe cultured broth and can thus be recovered by removing solid matter,including the mycelium, for example by filtration (preferably using afilter aid such as diatomaceous earth) or by centrifugation. It can thenbe recovered from the separated liquid portion by conventionaltechniques and, if desired, then purified.

Chloropolysporins B and C are preferably separated from other productsin said liquid portion by means of an adsorbent, either by adsorbing theimpurities or by adsorbing the chloropolysporins or by adsorbing bothseparately or together and then eluting the chloropolysporins. A widerange of adsorbents may be used; examples which we have found to beparticularly satisfactory include: activated carbon; and adsorbingresins such as Amberlite (registered trade mark) XAD-2, XAD-4 or XAD-7(products of Rohm and Haas), Diaion (registered trade mark) HP 10, HP20, CHP 20P or HP 50 (products of Mitsubishi Chemical Industries Co.,Ltd.) and polyamide gels (a product of Woelm Pharma, West Germany). Theimpurities present in the liquid portion may be removed by passing thesolution containing the chloropolysporins through a layer or column ofone or more of the aforementioned adsorbents or by adsorbing thechloropolysporins on one or more of the adsorbents and then eluting thechloropolysporins, either separately or together, with a suitableeluent. Suitable eluents include mixtures of methanol, acetone orbutanol with water.

The chloropolysporins B and C thus obtained may be further purified byvarious means. Suitable methods include: partition column chromatographyusing a cellulose product, such as Avicel (a registered trade mark for aproduct of Asahi Chemical Industry Co., Ltd.) or Sephadex LH-20 (aregistered trade mark for a product of Farmacia, Sweden); reverse phasecolumn chromatography using a carrier for the reverse phase; extractionbased on the differences in distribution in solvents betweenchloropolysporins B and C and their contaminating impurities; or thecounter-current distribution method. These purification techniques maybe used singly or in combination and may, if needed, be repeated one ormore times.

Chloropolysporins B and C are preferably separated from each other bychromatography. A preferred system for this purpose is System 500 (aproduct of Waters Co.), using the Preppack C₁₈ cartridge. A suitableeluent is a buffered mixture containing acetonitrile and maintained at aslightly acidic pH value.

Depending upon the culture conditions, chloropolysporins B and C canexist in the mycelium from the culture broth and can be extractedtherefrom by conventional techniques. For example, they can be extractedwith a hydrophilic organic solvent (such as an alcohol or acetone), andthen the solvent removed from the extract to leave a residue, which isdissolved in an aqueous medium. The chloropolysporins can be extractedfrom the resulting solution and purified as described above.

Chloropolysporins B and C thus obtained have, as their sulfates, thephysical and chemical properties described above. They are normally andpreferably separated from the culture broth in the form of awater-soluble salt and are most conveniently characterized in the formof such a salt, i.e., as herein, in the form of the sulfate, sincechloropolysporins B and C themselves (i.e. the free bases) are insolublein water.

Since chloropolysporins B and C are amphoteric in character, they formsalts and these salts also form part of the present invention. Thenature of such salts is not critical, except that, where they are to beused for medicinal or veterinary purposes, they must be medicinallyacceptable, i.e. they must not, or must not to a significant extent,either have increased toxicity or have reduced activity, as comparedwith the free base. Examples of suitable acids for the formation of suchsalts include: inorganic acids, such as hydrochloric acid, sulfuric acidor phosphoric acid; organic carboxylic acids, such as acetic acid,citric acid, tartaric acid, malonic acid, maleic acid, malic acid,fumaric acid, itaconic acid, citraconic acid or succinic acid; andorganic sulfonic acids, such as methanesulfonic acid, benzenesulfonicacid, naphthalenesulfonic acid or p-toluenesulfonic acid.

Where the chloropolysporin B or C is isolated in the form of a salt, itmay be converted to the free base by conventional means, such as the useof ion-exchange resins or of adsorbents for reverse phasechromatography. An aqueous solution of a salt will normally have anacidic pH value; adjustment of this pH value to approximate neutralitywill result in mainly precipitation of the free base, which may then becollected by suitable means, e.g. filtration or centrifugation. Thisproduct will, however, normally be contaminated by impurities, includingminor proportions of the relevant salts, and will, therefore, normallyrequire further purification. Accordingly, a more preferred method is byusing, for example, a suitable ion-exchange resin or an adsorbent forreverse phase chromatography. These compounds, however, share with knownglycopeptide antibiotics, such as avoparcin, the property of being verydifficult to isolate in the form of the free base [see e.g. W. J.McGahren et al., Journal of Antibiotics, XXXVI, 12, 1671 (1983)] andthey are, accordingly, preferably isolated and employed in salt form.

It is currently believed that chloropolysporins B and C may berepresented by the planar structural formula: ##STR1## in which:

for chloropolysporin B,

R¹ represents a ristosamine residue;

R² represents a mannose residue;

R³ represents a glucose residue; and

R⁴ represents a rhamnose residue;

for chloropolysporin C.,

R¹ represents a ristosamine residue;

R² represents a mannose residue;

R³ represents a glucose residue; and

R⁴ represents a hydrogen atom.

Accordingly, chloropolysporins B and C differ only in the substituentrepresented by --OR⁴ in the above formula.

It is probable that the various assymetric carbon atoms shown in theabove formula adopt, in chloropolysporins B and C, specificconfigurations, but these have not, to date, been elucidated.

The minimal inhibitory concentrations (MIC) of chloropolysporins B and Cagainst various gram-positive and gram-negative bacteria were determinedby the two-fold agar dilution method, using a Mueller-Hinton agar medium(produced by Difco); the MIC against anaerobic bacteria was determinedusing a GAM agar medium (produced by Nissui). The results are shown inTables 3 and 4.

                  TABLE 3                                                         ______________________________________                                                             MIC (μg/ml)                                                                Chloropolysporin                                         Test strain            B        C                                             ______________________________________                                        Staphylococcus aureus                                                                        FDA 209P JC-1                                                                             1.56     1.56                                      Staphylococcus aureus                                                                        SANK 70175  3.13     1.56                                      Staphylococcus aureus                                                                        Smith       12.5     6.25                                      Staphylococcus SANK 71575  3.13     3.13                                      epidermidis                                                                   Enterococcus faecalis                                                                        SANK 71778  1.56     1.56                                      Bacillus subtilis                                                                            PCI 219     0.78     0.78                                      Mycobacterium smegmatis                                                                      ATCC 607    25.0     12.5                                      Escherichia coli                                                                             NIHJ JC-2   >100     >100                                      Klebsiella pneumoniae                                                                        PCI 602     >100     >100                                      Pseudomonas aeruginosa                                                                       NCTC 10490  >100     >100                                      Serratia marcescens                                                                          SANK 73060  >100     >100                                      Proteus mirabilis                                                                            SANK 70461  >100     >100                                      ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                                            MIC (μg/ml)                                                                Chloropolysporin                                          Test strain           B       C                                               ______________________________________                                        Bacteroides fragilis  >100    >100                                            Eubacterium cylindroides                                                                            6.25    3.13                                            Fusobacterium necrophorum                                                                           >100    >100                                            Peptostreptococcus saccharoliticus                                                                  6.25    3.13                                            Peptostreptococcus parvulus                                                                         0.78    0.39                                            Propionibacterium acnes                                                                             0.78    0.39                                            Clostridium symbiosum 1.56    0.39                                            Clostridium ramosum   1.56    1.56                                            Clostridium perfringens                                                                             0.20    0.10                                            Clostridium difficile 0.78    0.39                                            ______________________________________                                    

From the results given in the above Tables, it can be seen thatchloropolysporins B and C are effective against aerobic gram-positivebacteria, such as Staphylococcus aureus, Staphylococcus epidermidis,Enterococcus faecalis, Bacillus subtilis and Mycobacterium smegmatis,and against anaerobic gram-positive bacteria, such as Eubacteriumcylindroides, Peptostreptococcus saccharoliticus, Propionibacteriumacnes, Clostridium symbiosum, Clostridium perfringens and Clostridiumdifficile.

Comparison of the properties, chemical, physical and biological, givenabove of chloropolysporins B and C with those of known antibiotics leadsto the conclusion that they belong to the class of glycopeptideantibiotics containing chlorine, such as vancomycin, a voparcin α and β,actinoidins A and B or A-35512 B. However, chloropolysporins B and C canbe clearly distinguished from these known antibiotics on the basis ofthe differences shown in the following Table 5. Specifically, they havedifferent neutral saccharide components and different amino acids areproduced on acid hydrolysis. Moreover, they move a different distance onhigh voltage paper electrophoresis (HVPE,3300 volts/60 cm, 1 hour, pH7.5, in 0.1M tris-hydrochloric acid buffer solution), and they havedifferent chlorine contents.

                  TABLE 5                                                         ______________________________________                                                                               Chlo-                                           Neutral                       rine                                            saccha-                       content                                Antibiotic                                                                             ride      Amino acid    Distance                                                                            (%)                                    ______________________________________                                        Vancomycin                                                                             Glucose   Aspartic acid       4.89                                                      N-methylleucine                                            Avoparcin α                                                                      Glucose,  4-Hydroxyphenyl-                                                                            9.4 cm                                                                              1.85                                            Mannose,  glycine,  .sub.--N-methyl-                                          Rhamnose   -p-hydroxyphenyl-                                                            glycine                                                    Avoparcin β                                                                       Glucose,  3-Chloro-4-hydroxy-                                                                         9.4 cm                                                                              3.65                                            Mannose,  phenylglycine,  .sub.--N-                                           Rhamnose  methyl- -p-hydroxy-                                                           phenylglycine                                              Actinoidin A                                                                           Glucose,  4-Hydroxyphenyl-    2.02                                            Mannose   glycine, Phenyl-                                                              alanine                                                    Actinoidin B                                                                           Glucose,  2-Chloro-3-hydroxy- 3.96                                            Mannose,  phenylglycine,                                                                Phenylalanine                                              A-35512B Glucose,                      1.82                                            Mannose,                                                                      Rhamnose,                                                                     Fucose                                                               Actaplanin                                                                             Glucose,                      1.96                                            Mannose                                                                       Rhamnose                                                             Ristocetin A                                                                           Glucose,                      0                                               Mannose                                                                       Rhamnose                                                                      Arabinose                                                            Ristocetin B                                                                           Glucose,                      0                                               Mannose,                                                                      Rhamnose                                                             Chloropoly-                                                                            Glucose,  3-Chloro-4-   4 cm  5.11                                   sporin B Mannose,  hydroxyphenyl-                                                      Rhamnose  glycine,  .sub.--N-methyl-                                                     -p-hydroxyphenyl-                                                            glycine                                                    Chloropoly-                                                                            Glucose,  3-Chloro-4-   4 cm  5.62                                   sporin C Mannose   hydroxyphenyl-                                                                glycine,  .sub.--N-                                                           methyl- -p-hydroxy-                                                           phenylglycine                                              ______________________________________                                    

The value reported above as "Distance" is the distance of movement onhigh voltage paper electrophoresis, measured using bioautography withBacillus subtilis PCI 219 as the test organism.

From the above findings, it can be seen that chloropolysporins B and Ccan be used as antibiotics against various diseases caused by bacterialinfections. The route of administration can vary widely and may beparenteral (e.g. by subcutaneous, intravenous or intramuscular injectionor by suppository) or oral (in which case it may be in the form of atablet, capsule, powder or granule). The dose will, of course, vary withthe nature of the disease to be treated, the age, condition and bodyweight of the patient and the route and time of administration; however,for an adult human patient, a daily dose of from 0.1 to 1.0 grams ispreferred and this may be administered in a single dose or in divideddoses.

Moreover, in view of the strong activity of chloropolysporins B and Cagainst infectious bacteria of the genus Clostridium, they can beexpected to be valuable growth-promoting agents for veterinary use.Bacteria of the genus Clostridium, particularly Clostridium perfringensand Clostridium difficile, are often present in the intestines of farmanimals and are the cause of diarrhoea. Since chloropolysporins B and Chave a strong activity against such microorganisms, they would suppressthe growth of such microorganisms in the intestines and thus improve themicrobial balance of the intestines. This, in turn, would improve feedefficiency, thus contributing to weight gain and improved milkproduction in various farm animals, including ruminants, pigs andpoultry. Moreover, chloropolysporins B and C, in common with the otherglycopeptide antibiotics, are likely to have a low rate of absorptionthrough the digestive organs and, as a result, where thechloropolysporin B or C is administered in the feed, little will remainin the animal body and hence in animal products, such meat, milk oreggs. When the chloropolysporin B or C is used as a growth-promotingagent for animals, it is preferably administered orally. Although it maybe formulated into an edible composition with any suitable carrier ordiluent, it is particularly convenient to administer it in admixturewith an animal feed or with drinking water. When the chloropolysporin Bor C is used as a feed additive, it may be mixed alone with the feed orit may be mixed in combination with other non-toxic edible excipients,nutrient supplements (e.g. vitamins, minerals or amino-acids), otherantibiotics, anticoccidial agents or enzymes. For administration toanimals as a growth-promoting agent, the chloropolysporin B or C neednot necessarily be in a completely purified form and it may be used in acrude or partially purified form, as obtained at any desired stageduring the extraction and purification described above. For use as agrowth-promoting agent, chloropolysporin B or C is preferably employedin an amount of from 1 to 200, more preferably from 5 to 60, ppm byweight on the basis of the feed, drinking water or other carrier towhich it is added; where an impure form of chloropolysporin B or C isemployed, a concentration having equivalent activity is used.

Animals to which chloropolysporin B or C can be administered includefarm mammals (e.g. cattle, horses, swine, sheep and goats), poultry(e.g. chickens, turkeys and ducks) and pet animals (e.g. dogs, cats andbirds). Most significantly, when chloropolysporin B or C is administeredorally to animals, their growth is effectively promoted, but it islittle absorbed from the gastro-intestinal tract and it exhibits lowretention in animal tissues: thus, there is an almost complete absenceof chloropolysporin B or C residues in the products (e.g. milk, meat oreggs) of animals to which it has been administered, which is a greatadvantage from the view point of food hygiene.

The invention is further illustrated by the following examples.

EXAMPLE 1 PREPARATION OF CHLOROPOLYSPORINS B AND C

One loopful growth of Micropolyspora sp. SANK 60983 was inoculated intoa 500 ml Erlenmeyer flask containing 80 ml of medium A, which has thefollowing composition (percentages are by weight):

    ______________________________________                                        MEDIUM A                                                                      ______________________________________                                        Glucose                  3%                                                   Pressed yeast            1%                                                   Soybean meal             3%                                                   Calcium carbonate        0.4%                                                 Magnesium sulfate        0.2%                                                 Anti-foaming agent (Nissan CB-442)                                                                     0.01%                                                Water                    the balance                                          (adjusted to pH 7.0)                                                          ______________________________________                                    

The microorganism was then cultured for 84 hours at 28° C., using arotary shaker at 220 r.p.m.

25 ml of the resulting seed culture were inoculated into each of four 2liter Erlenmeyer flasks, each containing 500 ml of medium B, which hasthe following composition. (percentages are by weight):

    ______________________________________                                        MEDIUM B                                                                      ______________________________________                                        Glucose                  5%                                                   Yeast extract            0.1%                                                 Soybean meal             1%                                                   Polypepton (a product of Daigo                                                                         0.4%                                                 Eiyo Co. Ltd., Japan)                                                         Beef extract             0.4%                                                 Sodium chloride          0.25%                                                Calcium carbonate        0.5%                                                 Anti-foaming agent (Nissan CB-442)                                                                     0.01%                                                Water                    the balance                                          (adjusted to pH 7.2)                                                          ______________________________________                                    

The microorganism was then cultured at 28° C. for 24 hours, using arotary shaker at 220 r.p.m.

The resulting cultured broths were combined. 750 ml of this broth werethen inoculated into each of two 30 liter jar fermentors, eachcontaining 15 liters of medium B, and the microorganism was thencultured at 28° C. for 69 hours, whilst aerating at the rate of 15liters per minute and stirring.

At the end of this time, batches of cultured broth separately cultivatedas described above were combined to give a total of 30 liters ofcultured broth. Celite 545 (a registered trade mark for a product ofJohns-Manville Products Corp, New Jersey, U.S.A.) filter aid was addedto the cultured broth and the mixture was filtered, to give 30 liters ofa filtrate. This filtrate was adsorbed on 3 liters of Diaion HP 20 (aproduct of Mitsubishi Chemical Industries Co., Ltd.), and the adsorbentwas washed with water and then eluted with 50% v/v aqueous acetone.Acetone was evaporated from the combined active fractions by evaporationunder reduced pressure; and the concentrate thus obtained waslyophilized, giving 44 g of a crude powder.

41 g of this powder were dissolved in water and adsorbed onto 1.8 litersof Diaion HP 20, washed with 5 liters of water and 2 liters of 10% v/vaqueous acetone, and then eluted with 4 liters of 50% v/v aqueousacetone. The active fractions from the elution were collected andcondensed to a volume of 1 liter by evaporation under reduced pressure.The condensate was centrifuged at 5000 r.p.m. and the resultingprecipitate was dried, to give 9.6 g of crude powder containingchloropolysporins B and C.

This crude powder was dissolved in 1 liter of 50% v/v aqueous methanoland then adsorbed onto 200 ml of acidic alumina (a product of WoelmPharma, West Germany), which had previously been equilibrated with 50%v/v aqueous methanol. The adsorbed product was then eluted with the samesolvent, and the active fractions, a total of 1.1 liters, werecollected. The combined active fractions were passed through 60 ml ofDowex 21 K (OH⁻), and eluted with water. The active fractions from thiselution, a total volume of 1.2 liters, were collected and then condensedby evaporation under reduced pressure to a volume of 30 ml. Thiscondensate was lyophilized, to give 1.23 g of powder. The powder wasdissolved in aqueous hydrochloric acid of pH 4.0 and then adsorbed onto56 g of Polyamide filled with water (a product of Woelm Pharma, WestGermany). This was subjected to gradient elution with 400 ml of waterand 1.2 liters of methanol, in 20 ml fractions, up to fraction 80.Fractions 30-60 were collected and combined. The methanol was distilledoff under reduced pressure and the resulting concentrate waslyophilized, to give 738 mg of a white powder containingchloropolysporins B and C.

4.4 g of this crude white powder containing chloropolysporins B and Cwere dissolved in 80 ml of a mixed solvent consisting of 15 parts ofacetonitrile and 85 parts of a buffer solution (containing 0.2% w/vsodium heptanesulfonate, 2.5% w/v acetic acid and 0.5% w/v concentratedaqueous ammonia); the solution was then adsorbed on a System 500chromatography system (a product of Waters Co), using a Preppack C₁₈cartridge. This was developed and eluted with the same mixed solvent asmentioned above at a flow rate of 100-150 ml per minute.Chloropolysporin B was eluted in the solvent after between 800 ml and1700 ml of the eluant had passed through the cartridge, whilstchloropolysporin C was eluted after between 1700 and 4700 ml of theeluant had passed.

The active fractions containing chloropolysporin B were collected andadjusted to a pH value of 7.0. They were then concentrated byevaporation under reduced pressure, to distill off the acetonitrile. Theresulting concentrated solution was adsorbed on a Diaion HP 20 column(100 ml), washed with water, and then eluted with 500 ml of 70% v/vaqueous acetone. The eluate was condensed by evaporation under reducedpressure, and the residue was lyophilized to afford chloropolysporin Bheptanesulfonate as a powder.

200 mg of this powder were dissolved in 5 ml of water, and then 1 ml ofa 10% w/v aqueous solution of sodium dodecylsulfate was dropped into theresulting solution. The precipitate which formed was collected bycentrifugation at 3000 rpm for 10 minutes. This precipitate wassuspended in water and the suspension was again centrifuged at 3000 rpmfor 10 minutes to wash the precipitate. This operation was repeated afurther three times to wash the precipitate. The precipitate was thendissolved in 3 ml of methanol and the insoluble residue was filteredoff. 2 ml of a 0.5M methanolic solution of triethylamine sulfate wereadded dropwise and the resulting precipitate was collected bycentrifugation at 3000 rpm for 10 minutes. This precipitate wassuspended in a small amount of methanol and again centrifuged at 3000rpm for 10 minutes. This was repeated a further three times to wash theprecipitate. The precipitate was then dissolved in 1.5 ml of water andthe insoluble residue was filtered off. Lyophilization of the filtrategave 65 mg of chloropolysporin B sulfate.

The active fractions containing the chloropolysporin C were collectedand adjusted to a pH value of 7.0. They were then concentrated byevaporation under reduced pressure to distill off the acetonitrile, andthe concentrate was absorbed on a Diaion HP 20 column (50 ml), washedwith water, and then eluted with 300 ml of 70% v/v aqueous acetone. Theeluate was condensed by evaporation under reduced pressure, and theresidue was lyophilized to afford 1.0 g of a powder containingchloropolysporin C heptanesulfonate. This powder was dissolved in 10 mlof a mixed solvent consisting of 15 parts of acetonitrile and 85 partsof a buffer solution (containing 0.2% w/v sodium heptanesulfonate, 2.5%w/v acetic acid and 0.5% w/v concentrated aqueous ammonia) and each 2 mlof the resulting solution was adsorbed on a Lobar column RP-18 (size B,a product of Merck and Co). This was developed and eluted at 13 ml perminute with the same mixed solvent, and the chloropolysporin C waseluted 30-40 minutes after commencement of the elution, whilst thecontaminating chloropolysporin B was eluted between 18 and 20 minutes.This operation was repeated a total of 5 times.

The chloropolysporin C fractions were collected and adjusted to a pHvalue of 5.8. They were then concentrated by evaporation under reducedpressure. The concentrate was adsorbed onto 40 ml of Diaion HP 20,washed with water and eluted with 200 ml of 50% v/v aqueous acetone. Theeluate was condensed by evaporation under reduced pressure, and theresidue was lyophilized, to give 250 mg of a crude chloropolysporinC-containing powder. This powder was dissolved in 5 ml of 50% v/vaqueous methanol, and the solution was adsorbed on 150 ml of ToyopearlHW40F (a product of Toyo Soda Co Ltd), which had previously beenequilibrated with 50% v/v aqueous methanol. The column was developed andeluted with 50% v/v aqueous methanol at a flow rate of 0.6 ml perminute. The eluate was collected in fractions of 2.5 ml each, and thechloropolysporin C heptanesulfonate was found to be present in fractions51-64.

These fractions were combined and then condensed under reduced pressure,and then 1 ml of a 10% w/v aqueous solution of sodium dodecylsulfate wasadded dropwise. The resulting precipitate was collected bycontrifugation at 3000 rpm for 10 minutes. The precipitate was thensuspended in water and washed by centrifugation at 3000 rpm for 10minutes. These operations were repeated a further three times. Theprecipitate was then dissolved in 3 ml of methanol and the insolubleresidue was filtered off. 2 ml of a 0.5M methanolic solution oftriethylamine sulfate were then added dropwise to the filtrate and theresulting precipitate was collected by centrifugation at 3000 rpm for 10minutes. This precipitate was suspended in methanol and washed bycentrifugation at 3000 rpm for 10 minutes. This operation was repeated afurther three times. The resulting precipitate was dissolved in 1.5 mlof water, the insoluble residue was removed, and the solution waslyophilized to afford 54 mg of chloropolysporin C sulfate.

The chloropolysporin B sulfate and chloropolysporin C sulfate obtainedas described above had the properties heretofor described.

EXAMPLE 2

1 loopful growth of Micropolyspora sp. SANK 60983 was inoculated into a500 ml Erlenmeyer flask containing 80 ml of medium A, having thecomposition described in Example 1. It was then cultivated at 28° C. for72 hours, using a rotary shaker at 220 rpm. 25 ml of this seed culturewere inoculated into each of six 2 liter Erlenmeyer flasks, eachcontaining 500 ml of medium B, having the composition described inExample 1, and cultivated for 24 hours at 28° C.

The whole of the cultured broth was then inoculated into a 100 litertank containing 60 liters of medium B and cultivated at 28° C. for afurther 24 hours.

15 liters of the resulting seed culture were then inoculated into eachof two 600 liter tanks, each containing 300 liters of medium B, and themicroorganism was cultivated, whilst stirring, for 67 hours at 28° C.,with aeration at the rate of 300 liters per minute, with an internalpressure of 1.0 kg/cm² and with 3-5 ppm of dissolved oxygen.

At the end of the cultivation period, a Celite 545 filter aid was addedto the culture and the culture was filtered, to afford 550 liters offiltrate. This filtrate was passed through a Diaion HP 20 column (60liters) to adsorb the chloropolysporin B. The column was washed withwater and eluted with 50% v/v aqueous acetone, yielding 570 liters ofactive fractions. The acetone was then distilled off under reducedpressure, leaving 280 liters of a concentrate. This concentrate wasextracted twice, each time with 200 liters of butanol to remove theimpurities, and the residual aqueous layer was condensed to 5 liters byevaporation under reduced pressure. The concentrated solution wasadjusted to a pH value of 5.8 by the addition of 1N aqueous sodiumhydroxide, and then the solution was adsorbed on a column containing 4.2liters of Polyamide gel (a product of Woelm Pharma, West Germany), anddeveloped and eluted with water. The eluate was separated in fractionsof 1 liter each, and chloropolysporin B was eluted in fractions 3-9.These fractions were combined, and adjusted to a pH value of 4.0 by theaddition of 1N aqueous hydrochloric acid. The resulting 7 liters ofactive fractions were condensed by evaporation under reduced pressure,and the residue was lyophilized, to give 42 g of chloropolysporin Bhydrochloride.

Elemental analysis: Calculated for C₈₃ H₈₉ O₃₄ N₈ Cl₃.0.5HCl.4H₂ O: C,51.41%; H, 5.07%; N, 5.78%; Cl, 6.40%. Found: C, 51.05%; H, 5.37%; N,6.36%; Cl, 6.90%.

Following the same procedure as described in Example 2, but employingthe chloropolysporin C-containing fractions, chloropolysporin Chydrochloride was also produced.

Elemental analysis: Calculated for C₇₇ H₇₉ O₃₀ N₈ Cl₃.HCl.9H₂ O: C,48.64%; H, 5.19%; N, 5.89%; Cl, 7.45%. Found: C, 48.48%; H, 5.13%; N,5.81%; Cl, 7.78%.

EXAMPLE 3 CAPSULES FOR ORAL USE

The following ingredients were mixed:

    ______________________________________                                        Chloropolysporin B hydrochloride                                                                       100 mg                                               Lactose                  100 mg                                               Corn starch              148.5 mg                                             Magnesium stearate       1.5 mg                                               ______________________________________                                    

The mixture was sieved through a 30 Tyler standard mesh sieve, giving350 mg of a fine powder, which was put into a No. 2 gelatin capsule.

EXAMPLE 4

The procedure described in Example 3 was repeated, except thatchloropolysporin C hydrochloride was employed in place ofchloropolysporin B hydrochloride.

We claim:
 1. An antibiotic substance named chloropolysporin C andpharmaceutically acceptable salts thereof, which, as its sulfate, ischaracterized by the following properties:(a) it takes the form of anamphoteric white powder, soluble in water; (b) specific rotation: [α]²⁵-64.4° (C=1.08, 0.1N aqueous hydrochloric acid, sodium D-line); (c)elemental analysis: C, 50.53%; H, 4.69%; N, 6.14%; Cl, 5.62%; S, 1.12%;(d) on acid hydrolysis it yields: neutral saccharides: glucose andmannose; amino acids: 3-chloro-4-hydroxyphenylglycine andN-methyl-p-hydroxyphenylglycine; (e) ultraviolet absorption spectrum: asillustrated in FIG. 4 of the accompanying drawings, having an absorptionmaximum λ_(max) at 280 nm (E_(1cm) =57) in a 0.1N solution ofhydrochloric acid, the absorbence, E, being measured at a concentrationof 1% w/v; (f) infrared absorption spectrum: the infrared absorptionspectrum (νcm⁻¹) measured on a KBr disc is as shown in FIG. 5 of theaccompanying drawings; (g) nuclear magnetic resonance spectrum: thenuclear magnetic resonance spectrum (δ ppm), measured at 400 MHz indeuterated dimethyl sulfoxide using tetramethylsilane as the internalstandard, is as illustrated in FIG. 6 of the accompanying drawings; (h)solubility: soluble in water and methanol, sparingly soluble in acetone,and insoluble in ethyl acetate, chloroform and benzene; (i) colorreactions: positive in Ninhydrin and Rydon-Smith reactions; (j) thinlayer chromatography: Rf value=0.65, using a cellulose sheet (Eastman)as adsorbent and a 15:10:3:12 by volume mixture of butanol, pyridine,acetic acid and water as the developing solvent; (k) molecular formula:C₇₇ H₇₉ O₃₀ N₈ Cl₃.0.5H₂ SO₄.5H₂ O; (l) molecular weight: the molecularweight, measured by FAB-MS, was 1700 (MH⁺, 1701).
 2. A compound of theformula: ##STR2## wherein: R¹ represents a ristosamine residue;R²represents a mannose residue; R³ represents a glucose residue; and R⁴represents a hydrogen atom;or a pharmaceutically acceptable saltthereof.
 3. A pharmaceutical or veterinary composition comprising theantibiotic substance chloropolysporin C or a salt thereof in anantibiotic effective amount as defined in claim 1 in admixture with apharmaceutical or veterinary carrier or diluent.
 4. An antibioticcomposition comprising 0.1 to 1.0 grams of the chloropolysporin C orsalt thereof of claim 1 in a pharmaceutical carrier or diluent.
 5. Amethod for the treatment or prophylaxis of bacterial infections byadministering an antibiotic effective amount the antibiotic substancechloropolysporin C or a salt thereof as defined in claim 1 to an animal.6. A method of promoting the growth of a farm animal by the oraladministration of an effective amount of the antibiotic substancechloropolysporin C or a salt thereof as defined in claim 1 to saidanimal.